Energy Coupling in H-Amino Acid Cotransport : ATP DEPENDENCE OF THE SPONTANEOUS ELECTRICAL REPOLARIZATION OF THE CELL MEMBRANES IN OAT COLEOPTILES

Experiments were undertaken in order to test the mechanism of energy coupling for amino acid uptake proposed in the cotransport hypothesis. According to the hypothesis an electrochemical potential difference in H⁺ is established by active H⁺ extrusion. That potential difference then drives the cotransport of H⁺ and amino acids into the cells. Application of amino acids to oat (Avena sativa var. Victory) coleoptiles induced transient depolarizations of the cell membrane electrical potentials considered to reflect the joint uptake of H⁺ and amino acids followed by an enhanced H⁺ extrusion. In the presence of KCN, cysteine induced strong depolarizations, but the rate of repolarization depended linearly upon the cyanide-adjusted ATP level of the tissue. At an ATP level 44% of normal, the membrane potential was 74% of normal, but the repolarization after cysteine-induced depolarization was practically nil. Sudden transitions from room temperature to temperatures below 15° C induced sharp depolarizations of the membrane which then repolarized within 3 min; the ATP content of the tissues was unaffected. Cysteine and alanine induced strong depolarizations at temperatures between 5 and 25°C, and the Q₁₀ for the rate of depolarization was 1.5 for cysteine and 1.6 for alanine. The Q₁₀ for the rate of repolarization was 3.0 for cysteine and 2.0 for alanine. These experiments support the prevailing view that the depolarizations are caused by the passive joint influx of H⁺ and amino acids and that the repolarizations depend upon the ATP-dependent extrusion of H⁺.     

Electrical evidence for rhythmic changes in the cotransport of sucrose and hydrogen ions in Samanea pulvini

Measurements with microelectrodes implanted into Samanea saman (Jacq.) Merrill leaf pulvini showed that membrane potentials were rhythmically sensitive to the application of sucrose. The magnitude of the electrical depolarizations induced by sucrose were dependent on the concentration of H⁺ in the medium, yet changes in [H⁺] alone did not greatly affect the potential. During sucrose-induced electrical depolarization, there was a slight increase in the pH of the bathing medium; both effects were abolished by high levels of K⁺, Na⁺ or Ca²⁺ in the medium. These observations indicate that H⁺ enter the cells by some cooperative action with sucrose. A model of H⁺-substrate cotransport is proposed in which a sugar carrier in the membrane is made more permeable by the attachment of a proton. The rhythmic nature of this proposed cotransport may be related to circadian leaf-movements in this plant.     

Electrical evidence for different mechanisms of uptake for basic, neutral, and acidic amino acids in oat coleoptiles

The application of neutral or acidic amino acids to oat coleptiles induced transient depolarizations of the membrane potentials. The depolarizations are considered to reflect H⁺ -amino acid co-transport, and the spontaneous repolarizations are believed to be caused by subsequent electrogenic H⁺ extrusion. The basic amino acids depolarized the cell membrane strongly, but the repolarizations were weak or absent. The depolarizations induced by the basic amino acids were weakly sensitive to manipulations of the extracellular and intracellular pH. The depolarizations induced by the other amino acids, in contrast, were more strongly affected by the pH changes. Several amino acids induced distinct but diminished depolarizations in the presence of 2,4-dinitrophenol or cyanide, but the repolarizations were generally eliminated. These experiments support the co-transport theory but suggest somewhat different mechanisms for the transport of the neutral, acidic, and basic amino acids. We suggest that the neutral amino acids are co-transported with a single H⁺ and that accumulation depends upon both the ΔpH and the membrane potential components of the proton motive force. The acidic amino acids appear to be accumulated by a similar mechanism except that the transport of each molecule may be associated with a cation in addition to a single proton. The permanently protonated basic amino acids appear not to be co-transported with an additional proton. Accumulation would depend only on the membrane potential component of the proton motive force.     

Extra- and Intracellular pH and Membrane Potential Changes Induced by K, Cl, H(2)PO(4), and NO(3) Uptake and Fusicoccin in Root Hairs of Limnobium stoloniferum

Short-term ion uptake into roots of Limnobium stoloniferum was followed extracellularly with ion selective macroelectrodes. Cytosolic or vacuolar pH, together with the electrical membrane potential, was recorded with microelectrodes both located in the same young root hair. At the onset of chloride, phosphate, and nitrate uptake the membrane potential transiently decreased by 50 to 100 millivolts. During Cl⁻ and H₂PO₄⁻ uptake cytosolic pH decreased by 0.2 to 0.3 pH units. Nitrate induced cytosolic alkalinization by 0.19 pH units, indicating rapid reduction. The extracellular medium alkalinized when anion uptake exceeded K⁺ uptake. During fusicoccin-dependent plasmalemma hyperpolarization, extracellular and cytosolic pH remained rather constant. Upon K⁺ absorption, FC intensified extracellular acidification and intracellular alkalinization (from 0.31 to 0.4 pH units). In the presence of Cl⁻ FC induced intracellular acidification. Since H⁺ fluxes per se do not change the pH, recorded pH changes only result from fluxes of the stronger ions. The extra- and intracellular pH changes, together with membrane depolarization, exclude mechanisms as K⁺/A⁻ symport or HCO₃⁻/A⁻ antiport for anion uptake. Though not suitable to reveal the actual H⁺/A⁻ stoichiometry, the results are consistent with an H⁺/A⁻ cotransport mechanism.     

Implications for cytoplasmic pH,protonmotice force,and amino-acid transport across the plasmalemma of Riccia fluitans

By means of pH-sensitive microelectrodes, cytoplasmic pH has been monitored continuously during amino-acid transport across the plasmalemma of Riccia fluitans rhizoid cells under various experimental conditions. (i) Contrary to the general assumption that import of amino acids (or hexoses) together with protons should lead to cytoplasmic acidification, an alkalinization of 0.1–0.3 pH_c units was found for all amino acids tested. Similar alkalinizations were recorded in the presence of hexoses and methylamine. No alkalinization occurred when the substrates were added in the depolarized state or in the presence of cyanide, where the electrogenic H⁺-pump is inhibited. (ii) After acidification of the cytoplasm by means of various concentrations of acetic acid, amino-acid transport is massively altered, although the protonmotive force remained essentially constant. It is suggested that H⁺-cotransport is energetically interconnected with the proton-export pump which is stimulated by the amino-acid-induced depolarization, thus causing proton depletion of the cytoplasm. It is concluded that, in order to investigate H⁺-dependent cotransport processes, the cytoplasmic pH must be measured and be under continuous experimental control; secondly, neither pH nor the protonmotive force across a membrane are reliable quantities for analysing a proton-dependent process.Abbreviations 3-OMG 3-oxymethylglucose - pH_c cytoplasmic pH - _m electrical potential difference across the respective membrane, i.e. membrane potential - _H+/F (=pmf) electrochemical proton gradient     

Proton/Phosphate Stoichiometry in Uptake of Inorganic Phosphate by Cultured Cells of Catharanthus roseus (L.) G. Don

Upon absorption of phosphate, cultured cells of Catharanthus roseus (L.) G. Don caused a rapid alkalinization of the medium in which they were suspended. The alkalinization continued until the added phosphate was completely exhausted from the medium, at which time the pH of the medium started to drop sharply toward the original pH value. Phosphate exposure caused the pH of the medium to increase from pH 3.5 to values as high as 5.8, while the rate of phosphate uptake was constant throughout (10-17 micromoles per hour per gram fresh weight). This indicates that no apparent pH optimum exists for the phosphate uptake by the cultured cells. The amount of protons cotransported with phosphate was calculated from the observed pH change up to the maximum alkalinization and the titration curve of the cell suspension. Proton/phosphate transport stoichiometry ranged from less than unity to 4 according to the amount of phosphate applied. At low phosphate doses, the stoichiometries were close to 4, while at high phosphate doses, smaller stoichiometries were observed. This suggests that, at high phosphate doses, activation of the proton pump is induced by the longer lasting proton influx acidifying the cytoplasm. The increased H⁺ efflux due to the proton pump could partially compensate protons taken up via the proton-phosphate cotransport system. Thus, the H⁺/H₂PO₄⁻ stoichiometry of the cotransport is most likely to be 4.     

Evidence for a specific glutamate/h cotransport in isolated mesophyll cells

Mechanically isolated Asparagus sprengeri Regel mesophyll cells were suspended in 1 millimolar CaSO₄. Immediate alkalinization of the medium occured on the addition of 1 millimolar concentrations of l-glutamate (Glu) and its analog l-methionine-d,l-sulfoximine (l-MSO). d-Glu and the l isomers of the protein amino acids did not elicit alkalinization. l-Glu dependent alkalinization was transient and acidification resumed after approximately 30 to 45 minutes. At pH 6.0, 5 millimolar l-Glu stimulated initial rates of alkalinization that varied between 1.3 to 4.1 nmol H⁺/10⁶ cells·minute. l-Glu dependent alkalinization was saturable, increased with decreasing pH, was inhibited by carbonyl cyanide-p-trichloromethoxyphenyl hydrazone (CCCP), and was not stimulated by light. Uptake of l-[U-¹⁴C]glutamate increased as the pH decreased from 6.5 to 5.5, and was inhibited by l-MSO. l-Glu had no influence on K⁺ efflux. Although evidence for multiple amino acid/proton cotransport systems has been found in other tissues, the present report indicates that a highly specific l-Glu/proton uptake process is present in Asparagus mesophyll cells.     

pH and membrane-potential changes during glucose uptake inLemna gibba G1 and their response to light

Extracellular pH was measured with a microelectrode positioned over the lower surface of singleLemna gibba plants. Upon addition of glucose, a transient extracellular alkalinization occurred. Saturated extracellular pH changes were observed with 5 mM glucose. Simultaneously, the membrane potential difference of –250 mV in the dark measured with intracellular glass micropipettes, trnasiently decreased by 105 mV. Uptake of [¹⁴C]glucose and extracellular alkalinization was enhanced by light whereas glucose-induced membrane-potential changes were reduced in the light and became even smaller with increasing the preillumination time. Glucose uptake was optimal at pH 6. The results are taken as further evidence in favor of H⁺-glucose cotransport inLemna.Dedicated to Professor W. Simonis on the occasion of his 70th birthdayUniversity of Missouri Agricultural Experiment Station Journal Series, paper No. 8266     

H extrusion and potassium uptake associated with potential hyperpolarization in maize and wheat root segments treated with permeant weak acids

The rapid uptake of weak acids permeant in the uncharged form is accompanied in maize and wheat root segments by a hyperpolarization of the transmembrane electrical potential and an increase in K⁺ uptake, suggesting a stimulation of the plasmalemma H⁺ pump. The evaluation of weak acid-induced H⁺ extrusion must take into account the alkalinization of the medium due to the rapid uptake of the uncharged form of the acid, partially masking the proton pump-mediated extrusion of H⁺. The data corrected for this interference show that the lipophilic butyric acid and trimethyl acetic acid induce in maize and in wheat root segments a significant increase in `real' H⁺ extrusion, roughly matching the increase in net K⁺ uptake. The presence of K⁺ significantly increases the rate of uptake of the weak acid, possibly as a consequence of an alkalinization of the cytosol associated with K⁺ absorption. In maize root segments, the effects of fusicoccin and those of butyric acid on both K⁺ uptake and H⁺ extrusion are clearly synergistic, thus suggesting distinct modes of action. These results support the view that the activity of the plasmalemma H⁺ pump is regulated by the value of cytosolic pH.     

Membrane Potential and Proton Cotransport of Alanine and Phosphate as Affected by Permeant Weak Acids in Lemna gibba

The treatment of Lemna gibba plants with the weak acids (trimethylacetic acid and butyric acid), used as tools to decrease intracellular pH, induced a hyperpolarization of membrane potential, dependent on the concentration of the undissociated permeant form of the weak acid and on the value of the resting potential. Measurements were carried out both with `high potential' and `low potential' plants and the maximum values af acid induced hyperpolarizations were about 35 and 71 millivolts, respectively. Weak acids influenced also the transient light-dark membrane potential changes, typical for photosynthesizing material, suggesting a dependence of these changes on an acidification of cytoplasm. In the presence of the weak acids, the membrane depolarization induced by the cotransport of alanine and phosphate with protons was reduced; the maximum reduction (about 90%) was obtained with alanine during 2 millimolar trimethylacetic acid perfusion at pH 5. A strong inhibition of the uptake rates (up to 48% for [¹⁴C]alanine and 68% for ³²P-phosphate) was obtained in the presence of the weak acids, both by decreasing the pH of the medium and by increasing the concentration of the acid. In these experimental conditions, the ATP level and O₂ uptake rates did not change significantly. These results constitute good evidence that H⁺/solute cotransport in Lemna, already known to be dependent on the electrochemical potential difference for protons, is also strongly regulated by the cytoplasmic pH value.     

Relationship between Energy-dependent Phosphate Uptake and the Electrical Membrane Potential in Lemna gibba G1

High rates of phosphate uptake into phosphate-starved Lemna gibba L. G1 were correlated with a high membrane potential (pd = −220 millivolts). In plants maintaining a low pd (−110 millivolts), the uptake rate was only 20% of that of high-pd plants. At the onset of phosphate transport, the membrane of high-pd plants was transiently depolarized. This effect was much smaller in low-pd plants. Light stimulated phosphate uptake and the repolarization upon phosphate-induced depolarization, especially in plants grown without sucrose. The phosphate uptake rate was optimal at pH 6 and decreased with increasing pH, corresponding to the phosphate-induced pd changes. Phosphate starvation stimulated the uptake and increased the phosphate-induced depolarization, thus indicating that phosphate uptake depends on the intracellular phosphate level. It is suggested that uptake of monovalent phosphate in Lemna gibba proceeds by an H⁺ cotransport dependent on the proton electrochemical potential difference and, hence, on the activity of an H⁺ -extrusion pump.     

Suitability of Arabidopsis for studies on intracellular pH regulation: correlation between H+ pump activity, cytosolic pH and malate level in wild-type and in a mutant partially insensitive to fusicoccin

Data are presented on the suitability of Arabidopsis thaliana seedlings for studies on intracellular pH regulation. In this material, grown in the dark in liquid medium, the determination of weak acid distribution at equilibrium provides an adequate method for calculating cytosolic pH values, in spite of the failure of benzylamine as a vacuolar pH probe. The stimulation of the H⁺ pump by K⁺ or K⁺ and fusicoccin (FC) is associated with a marked alkalinization of both cytosol and cell sap, and with a strong increase in malate level, whereas its inhibition by erythrosin B (EB) leads to the opposite effects. A good quantitative correlation is evident between the changes in net H⁺ extrusion and those in intracellular pH and malate content, in particular, with FC+K⁺. Cell sap buffer capacity is strongly influenced by the different treatments, its changes being substantially accounted for by changes in malate level. A comparison between the values of intracellular pH and malate level in wt and in the 5-2 mutant shows that in the mutant the cytosolic pH is always more acidic, and the intracellular alkalinization induced by FC+K⁺ and also by K⁺ alone is significatively lower. These results support the view that the partial insensitivity of 5-2 to FC is due to a reduced functionality of the H⁺-extruding system on which FC acts, and that the depression of the H⁺ pump activity in the mutant does not depend on a possible regulation by constitutively higher cytosolic pH values.     

High affinity k uptake in maize roots: a lack of coupling with h efflux

We report here on the putative coupling between a high affinity K⁺ uptake system which operates at low external K⁺ concentrations (K_m = 10-20 micromolar), and H⁺ efflux in roots of intact, low-salt-grown maize plants. An experimental approach combining electrophysiological measurements, quantification of unidirectional K⁺(⁸⁶Rb⁺) influx, and the simultaneous measurement of net K⁺ and H⁺ fluxes associated with individual cells at the root surface with K⁺- and H⁺-selective microelectrodes was utilized. A microelectrode system described previously (IA Newman, LV Kochian, MA Grusak, and WJ Lucas [1987] Plant Physiol 84: 1177-1184) was used to quantify net ion fluxes from the measurement of electrochemical potential gradients for K⁺ and H⁺ ions within the unstirred layer at the root surface. No evidence for coupling between K⁺ uptake and H⁺ efflux could be found based on: (a) extremely variable K⁺:H⁺ flux stoichiometries, with K⁺ uptake often well in excess of H⁺ efflux; (b) dramatic time-dependent variability in H⁺ extrusion when both fluxes were measured at a particular location along the root over time; and (c) a lack of pH sensitivity by the high affinity K⁺ uptake system (to changes in external pH) when net K⁺ uptake, unidirectional K⁺(⁸⁶Rb⁺) influx, and K⁺-induced depolarizations of the membrane potential were determined in uptake solutions buffered at pH values from pH 4 to 8. Based on the results presented here, we propose that high affinity active K⁺ absorption into maize root cells is not mediated by a K⁺/H⁺ exchange mechanism. Instead, it is either due to the operation of a K⁺-H⁺ cotransport system, as has been hypothesized for Neurospora, or based on the striking lack of sensitivity to changes in extracellular pH, uptake could be mediated by a K⁺-ATPase as reported for Escherichia coli and Saccharomyces.     

Control of Cell pH in the T84 Colon Cell Line

Cell pH regulation was investigated in the T84 cell line derived from epithelial colon cancer. Cell pH was measured by ratiometric fluorescence microscopy using the fluorescent probe BCECF. Basal pH was 7.17 ± 0.023 (n= 48) in HEPES Ringer. After acidification by an ammonium pulse, cell pH recovered toward normal at a rate of 0.13 ± 0.011 pH units/min in the presence of Na⁺, but in the absence of this ion or after treatment with 0.1 mm hexamethylene amiloride (HMA) no significant recovery was observed, indicating absence of Na⁺ independent H⁺ transport mechanisms in HEPES Ringer. In CO₂/HCO⁻ ₃ Ringer, basal cell pH was 7.21 ± 0.020 (n= 35). Changing to HEPES Ringer, a marked alkalinization was observed due to loss of CO₂, followed by return to the initial pH at a rate of −0.14 ± 0.012 (n= 8) pH/min; this return was retarded or abolished in the absence of Cl⁻ or after addition of 0.2 mm DIDS, suggesting extrusion of bicarbonate by Cl⁻/HCO⁻ ₃ exchange. This exchange was not Na⁺ dependent. When Na⁺ was added to cells incubated in 0 Na⁺ Ringer while blocking Na⁺/H⁺ exchange by HMA, cell alkalinization by 0.19 ± 0.04 (n= 11) pH units was observed, suggesting the presence of Na⁺/HCO⁻ ₃ cotransport carrying HCO⁻ ₃ into these cells, which was abolished by DIDS. These experiments, thus, show that Na⁺/H⁺ and Cl⁻/HCO⁻ ₃ exchange and Na⁺/HCO⁻ ₃ cotransport participate in cell pH regulation in T84 cells. Received: 3 April 2000/Revised: 22 June 2000     

Modulation of the membrane potential of endothelial cells from the guinea pig aorta by intracellular alkalinization

Endothelium-dependent vasoactive substances are known to evoke complex changes in the endothelial membrane potential (MP) and to increase intracellular pH in endothelial cells (EC). In our present study, we investigated the effect of agents able to increase intracellular pH on the MP of intact guinea pig aortic EC, and also the effect of blocking of Na⁺−H⁺ exchanger on ATP-induced electrical responses. Intracellular alkalinization was induced either by addition of ammonium chloride (NH₄Cl) to the superfusate, or by changing the bath solution saturated with 10% CO₂+90% O₂ to a solution saturated with 100% O₂. Both approaches evoked hyperpolarization of EC. After intracellular Ca²⁺ chelation by pretreatment of aortic preparations with 20 μM BAPTA-AM, the amplitude of NH₄Cl-induced hyperpolarization dropped from 3.9±0.6 to 0.7±0.3 mV. After pretreatment with ATP, NH₄Cl-induced hyperpolarization was not abolished, whereas after caffeine pretreatment this hyperpolarization was not observed. In the Na⁺-free solution and in the presence of furosemide, ATP-evoked hyperpolarization became longer. The same effect was also observed in the presence of sodium acetate, which directly acidifies the cytosol. In the Ca²⁺-free solution, furosemide did not induce prolongation of ATP-evoked hyperpolarization. Taking into account the results, it could be proposed that, first, hyperpolarization of EC after intracellular alkalinization is a result of Ca²⁺ release from the intracellular stores sensitive both to an increase in intracellular pH and to caffeine application. Second, intracellular alkalinization, being a result of activation of Na⁺−H⁺-antiporter, inhibits influx of extracellular Ca²⁺ into EC under ATP stimulation.     

Active oxygen species production in tobacco cells elicited by cryptogein*

We analyse the relationship between active oxygen species (AOS) production and pH changes induced in tobacco cells by cryptogein, a fungal proteinaceous elicitor of defence mechanisms in plants. When tobacco cells were treated with cryptogein, an intracellular acidification, an alkalinization of the extracellular medium and a transient burst of AOS (H₂O₂) were observed. Treatment of elicited cells with either diphenyleneiodonium (DPI), an inhibitor of the neutrophil NADPH oxidase, or Tiron, which scavenges O₂^˙? abolished AOS production. These data suggest the involvement of a NADPH oxidase-like enzyme leading to H₂O₂ production through O₂^˙? dismutation. Although H₂O₂ production could be, per se, the origin of the pH changes observed, we showed that it was not the main cause, since DPI and Tiron did not inhibit extracellular alkalinization. On the other hand, cryptogein-induced changes in pH could be abolished using fusicoccin (FC), which is known to stimulate the plasmalemma H⁺ ATPase. Consequently, the observed changes in pH induced by cryptogein could be mainly due to the inhibition of the plasmalemma H⁺-ATPase activity. Furthermore, changes in extracellular pH were shown to modulate the intensity of AOS production by elicited cells. The possible regulation of the NAD(P)H oxidase activity of plant cells by changes in pH is further discussed.     

Effect of K and H on sodium/citrate cotransport in renal brush-border vesicles

The uptake of citrate by renal brush-border vesicles, prepared according to the method of Vannier, occurs by Na⁺-linked cotransport. It is ‘positive rheogenic’, i.e., stimulated by an (inside) negative, and inhibited by an (inside) positive electrical potential. The question arises whether, besides Na⁺, other ions (e.g., K⁺ and H⁺) participate in the cotransport. As to K⁺, neither an inward nor an outward directed K⁺ gradient has a significant effect on the citrate movement, but at equal concentrations of K⁺ inside and outside, equilibrium exchange of citrate, and to a smaller extent, the Na⁺-linked net uptake of citrate, are significantly stimulated. This observation is consistent with a hypothetical model in which K⁺ acts by accelerating both the empty and the fully loaded translocator. As to H⁺, citrate uptake is also stimulated by decreasing extravesicular pH, an effect previously attributed to protonization of the citrate anion in the assumption that the resulting secondary citrate anion is more acceptable to the translocator site. It was found, however, that the pH effect is still apparent if the concentration of the secondary citrate is kept constant by adjusting the total citrate concentration. This is taken as an argument against the above assumption and as being consistent with H⁺-linked cotransport. After the overshoot peak citrate exits slowly, and even after several hours does not attain equilibrium distribution, presumably owing to trapping by vesicular calcium.     

Sucrose and proton cotransport in Ricinus cotyledons

In Ricinus cotyledons, evidence for proton extrusion came from observation of direct acidification of the medium in the presence of potassium salts. Increasing K⁺ influx with increasing pH suggested a link between K⁺ influx and H⁺ efflux by an H⁺ pump. The kinetics of K⁺ influx and H⁺ efflux were consistent with a 1:1 stoichiometry K⁺:H⁺, which may indicate either electrical coupling or carrier mediated exchange. The results were consistent with an H⁺ pump setting up an electrochemical potential gradient which provides the driving force for an H⁺-sucrose cotransport and the movement of K⁺. With reference to this, a model for phloem loading is suggested.     

Abscisic acid effects on intracellular pH and ion fluxes in barley leaf segments

Changes in intracellular pH and in H⁺, K⁺ and Cl^? fluxes were evaluated in different experimental conditions in leaf segments of barley (Hordeum vulgare cv. Georgie) incubated in the dark, at pH 5.5, in the presence or absence of abscisic acid (ABA), and a comparison was made between the effects of ABA and those of erythrosin B (EB), a plasmalemma H⁺-pump inhibitor. In all conditions tested, ABA induced a cell sap acidification, an alkalinization of the external medium, a decrease in K⁺ intracellular contents, and an increase in the contents of Cl^?. The ABA-induced decrease in K⁺ content was chiefly due to the inhibition of K⁺ influx. On the contrary, ABA did not influence the uptake of Cl^?, but inhibited Cl^? efflux, the inhibition satisfactorily accounting for the larger Cl^? content observed in the presence of the hormone. The intracellular acidification and the decrease in apparent outward net transport of H⁺ observed with ABA were seemingly not associated with the activity of the proton pump, the transmembrane electrical potential difference, or K⁺ transport. On the contrary, a correlation was evident with the changes in Cl^? content. These results and, in particular, the similarity between the effects of ABA and those induced by 4,4 -diisothiocyano-2,2-disulfonic acid stilbene (DIDS), a Cl^? channel-blocking agent, suggest that the ABA-induced changes in intracellular pH and in H⁺ transport might depend on the capability of ABA to inhibit Cl^? efflux, more than on a primary inhibition of the H⁺ pump, and propose an important role for ABA in regulating the Cl^? channels.     

The glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: Transport mechanism, regulation by ATP and characterization of the glutamine/glutamate antiport

The glutamine/amino acid transporter solubilized from rat renal apical plasma membrane (brush-border membrane) with C₁₂E₈ and reconstituted into liposomes has been previously identified as the ASCT2 transporter. The reconstituted transporter catalyses an antiport reaction in which external glutamine and Na⁺ are cotransported in exchange with internal glutamine (or other amino acids). The glutamine-Na⁺ cotransport occurred with a 1:1 stoichiometry. The concentration of Na⁺ did not influence the Km for glutamine and vice versa. Experimental data obtained by a bi-substrate analysis of the glutamine-Na⁺ cotransport, together with previous report on the glutamine_ex/glutamine_in pseudo bi-reactant analysis, indicated that the transporter catalyses a three-substrate transport reaction with a random simultaneous mechanism. The presence of ATP in the internal compartment of the proteoliposomes led to an increase of the Vmax of the transport and to a decrease of the Km of the transporter for external Na⁺. The reconstituted glutamine/amino acid transporter was inhibited by glutamate; the inhibition was more pronounced at acidic pH. A kinetic analysis revealed that the inhibition was competitive with respect to glutamine. Glutamate was also transported in exchange with glutamine. The external Km of the transporter for glutamate (13.3 mM) was slightly higher than the internal one (8.3 mM). At acidic pH the external but not the internal Km decreased. According with the Km values, glutamate should be transported preferentially from inside to outside in exchange for external glutamine and Na⁺.